A key event in the pathogenesis of Alzheimer's disease (AD) is the accumulation of amyloid-Beta peptide (ABeta), the principal protein component of the cerebral plaques characteristic of this disease. Two enzymes are involved in generating ABeta from the Amyloid Precursor Protein (APP): BACE, an extracellular aspartyl protease, generates the N-terminus; the enzymatic complex called gamma-secretase catalyzes the intramembrane proteolysis step resulting in the formation of the divergent C-termini of ABeta40 and ABeta42. Presenilin (PS) proteins, discovered as mutated loci in familial forms of AD, contain the catalytic aspartyl residues of gamma-secretase. While no potent inhibitors have been developed against BACE, several potent gamma-secretase inhibitors are available. Inhibition of gamma-secretase is thought to be key to successful therapeutic approaches; however, APP is not its only substrate. Several other Type I membrane proteins undergo gamma-secretase-dependent proteolysis within their putative membrane spanning segments and of these, the importance of proteolysis for Notch function is well established. Notch signaling plays an important role in human health. Intramembrane proteolysis of Notch is an essential regulated component of its signaling mechanism. Under saturating conditions, APP and Notch compete with each other for intramembrane proteolysis, but not for binding to PS, suggesting that multiple and perhaps distinct substrate binding sites exist on gamma-secretase. Since inhibition of gamma-secretase holds great promise for the treatment of Alzheimer's disease, it is essential we understand how gamma-secretase specifically recognizes its substrates. This will enable us to design inhibitors that can potentially block ABeta production by interfering with APP/PS interactions while avoiding interference with the cleavage of other substrates such as Notch. If one had access to crystallographic information, it would be possible to predict which amino acid substitutions would disrupt specific enzyme-substrate interfaces while sparing other important enzyme-substrate interfaces; however, one would still have to carry out empirical analyses for each pair of such interactions - a laborious and lengthy process that is unlikely to happen in the immediate future. We propose structure-activity approach that will shed light on the intramolecular and intermolecular interactions within gamma-secretase and between gamma-secretase and its substrates thus providing invaluable information; there is no good high-resolution structure of these interfaces to date. The genetic approach we propose is based on functional complementation assays; we will also conduct HTS screens in a novel assay directly measuring substrate-enzyme interactions.